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Published June 6, 2003 | Published
Journal Article Open

Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance, MscL - Implications for channel gating and evolutionary design

Abstract

Random mutagenesis of the mechanosensitive channel of large conductance (MscL) from Escherichia coli coupled with a high-throughput functional screen has provided new insights into channel structure and function. Complementary interactions of conserved residues proposed in a computational model for gating have been evaluated, and important functional regions of the channel have been identified. Mutational analysis shows that the proposed S1 helix, despite having several highly conserved residues, can be heavily mutated without significantly altering channel function. The pattern of mutations that make MscL more difficult to gate suggests that MscL senses tension with residues located near the lipid headgroups of the bilayer. The range of phenotypical changes seen has implications for a proposed model for the evolutionary origin of mechanosensitive channels.

Additional Information

© 2003 the American Society for Biochemistry and Molecular Biology. Received for publication, March 21, 2003, and in revised form, April 1, 2003. Published, JBC Papers in Press, April 1, 2003, DOI 10.1074/jbc.M302892200. Ann Batiza and Ching Kung, from the University of Wisconsin at Madison, generously provided wild type Ec-MscL in pB10b and Ian Booth, from the University of Aberdeen, generously provided the MJF465 bacterial strain. We are extremely grateful to Donald Elmore and the rest of the Dougherty group for insightful suggestions and to Prof. Henry Lester, Prof. Douglas Rees, Gerd Kochendoerfer, and their groups for additional helpful discussions.

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August 22, 2023
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